Abstract 1773: Overcoming paclitaxel resistance in triple-negative breast cancer using a novel barcoding technology
Authors: Milos Spasic, Qiuchen Guo, Adam Maynard, Gregory Goreczny, Adrienne Waks, Sara Tolaney, Elizabeth Mittendorf, Sandra McAllister
Journal: Cancer Research (2022)

Abstract Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype for which chemotherapy remains a part of standard treatment. Although pathologic complete response (pCR) after neoadjuvant chemotherapy, including paclitaxel (PTX), is associated with good outcomes, 50-60% of TNBC patients do not experience pCR and suffer poor long-term outcomes, often due to chemotherapy resistance. Identifying and analyzing the tumor cells responsible for chemotherapy resistance will lead to improved treatment strategies. We developed a model of TNBC resistance to neoadjuvant PTX using Met1 murine mammary carcinoma cells. To study clonal dynamics in response to PTX we developed a barcoding method, SunCatcher. First, 31 single-cell derived clonal populations were generated from parental Met1 cells. Each clone was infected with a lentiviral vector containing a unique DNA barcode detectable by qPCR. All barcoded clones (BCs) were mixed in equivalent numbers to generate a BC pool that we confirmed captures the parental Met1 heterogeneity and tumor growth kinetics. First, PTX responses were tested in vitro. The BC pool had an IC50 of ~100 nM, while individual BCs ranged in IC50 from 5 nM to 25 µM. We maintained the BC pool in 100 nM PTX for 39 days (termed long-term PTX; LTP), at which point it became clonal for BC25, suggesting that BC25 (IC50 of 100 nM) is uniquely PTX resistant, despite being the most proliferative and Zeb1hi/EpCAMlow clone in vitro. BC25 was not detected in the control-treated BC pool at 39 days. Next, the BC pool was orthotopically injected into FVB/NJ mice and treated the mice with 20 mg/kg PTX on days 7, 11, and 15. At the day 18 experimental endpoint, tumor volume significantly decreased by 56% in response to PTX compared to control. BC25 composition increased from 7.7% (controls) to 15% (PTX treated) within the tumors at endpoint. We also injected BC25 and LTP cells alone and administered the same PTX regimen once tumors reached 50 mm3. Both BC25 and LTP tumors were unresponsive to PTX and had a longer latency period (35d) than BC pool (10d). We performed a drug screen of 2313 compounds spanning FDA-approved cancer therapeutics on the LTP cells to identify compounds to target the PTX-resistant clone. HDAC inhibitors were the most potent class of hits and one, Panobinostat, killed LTP cells with an IC50 of 4.3 nM. Utilizing SunCatcher, we identified a unique PTX-resistant TNBC subclone that represents residual disease associated with poor long-term outcome. Typical in vivo experiments would have reached ethical endpoint before BC25 had a chance to grow, given its long latency, therefore this PTX-resistant clone would not have been identified. This is an important finding because therapeutic resistance can emerge after a protracted period, longer than typical pre-clinical experiments. Further work will explore mechanisms of resistance and the potential for combination therapies to prevent recurrent disease. Citation Format: Milos Spasic, Qiuchen Guo, Adam Maynard, Gregory Goreczny, Adrienne Waks, Sara Tolaney, Elizabeth Mittendorf, Sandra McAllister. Overcoming paclitaxel resistance in triple-negative breast cancer using a novel barcoding technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1773.